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Yeast Yeast 2005; 22: 1155–1169. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1308

Yeast Functional Analysis Report

Global metabolite analysis of yeast: evaluation of sample preparation methods
˚ ˆ Silas G. Villas-Boas§# , Jesper Højer-Pedersen§ , Mats Akesson† , Jørn Smedsgaard and Jens Nielsen*
Centre for Microbial Biotechnology, BioCentrum-DTU,Technical University of Denmark, Building 223, DK-2800 Kgs. Lyngby, Denmark

*Correspondence to: Jens Nielsen, Centre for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Building 223, DK-2800 Kgs, Lyngby, Denmark. E-mail: jn@biocentrum.dtu.dk authors contributed equally to this work.
# Present address: AgResearch Ltd, Grasslands Research Centre, Tennent Drive, PrivateBag 11008, Palmerston North, New Zealand. † Present address: BioProcess Laboratories, Novo Nordisk A/S, Novo All´ , DK-2880 Bagsvaerd, e Denmark. § Both

Abstract
Sample preparation is considered one of the limiting steps in microbial metabolome analysis. Eukaryotes and prokaryotes behave very differently during the several steps of classical sample preparation methods for analysis ofmetabolites. Even within the eukaryote kingdom there is a vast diversity of cell structures that make it imprudent to blindly adopt protocols that were designed for a specific group of microorganisms. We have therefore reviewed and evaluated the whole sample preparation procedures for analysis of yeast metabolites. Our focus has been on the current needs in metabolome analysis, which is the analysis of alarge number of metabolites with very diverse chemical and physical properties. This work reports the leakage of intracellular metabolites observed during quenching yeast cells with cold methanol solution, the efficacy of six different methods for the extraction of intracellular metabolites, and the losses noticed during sample concentration by lyophilization and solvent evaporation. A more reliableprocedure is suggested for quenching yeast cells with cold methanol solution, followed by extraction of intracellular metabolites by pure methanol. The method can be combined with reduced pressure solvent evaporation and therefore represents an attractive sample preparation procedure for high-throughput metabolome analysis of yeasts. Copyright  2005 John Wiley & Sons, Ltd. Keywords: extraction;metabolome; metabolomics; quenching; sample preparation; freeze-drying

Received: 20 October 2004 Accepted: 30 August 2005

Introduction
Metabolomics and metabolome analysis is a broadly used term for analysis of a large number of metabolites from a single organism. In metabolome analysis of microbial cells the separation of intraand extracellular metabolites is often required to gain insightinto the control of metabolic pathways. Therefore, it is important to determine the intracellular levels of metabolites accurately, which requires efficient and reliable methods for sample preparation. There have been considerable efforts to develop sensitive and accurate methods for detection of metabolites (Buchholz et al., 2002; Soga et al., 2002; van Dam et al., 2002; Mashego et al., 2004;Villas-Bˆ as et al., 2005; and others) o and to improve the efficiency of data mining
Copyright  2005 John Wiley & Sons, Ltd.

and data analysis (Goodacre et al., 2004; Kell, 2004; van der Greef et al., 2004) but relative little attention has been given to sample preparation procedures. Figure 1 summarizes the main steps in sample preparation for metabolome analysis. Since the metabolite levelsreflect the ultimate response of a biological system to genetic or environmental changes, rapid stopping (quenching) of the cell metabolism and extracellular enzymatic activity is the first step for such methods. Metabolite concentrations are very prone to changes induced by (unnoticed) variation in the environment of the cell (de Koning and van Dam, 1992). The quenching procedure aims to stop...
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